Supported by grants from the Swiss National Science Foundation(Grant 3200-037435.93) and from the Central Laboratory, BloodTransfusion Service, Swiss Red Cross

M. FurlanCentral Hematology Laboratory, Inselspital, University of Bern,CH-3010 Bern, Switzerland

Ann Hematol (1996) 72 :341–348 Q Springer-Verlag 1996

REVIEW ARTICLE

M. Furlan

Von Willebrand factor: molecular size and functional activity

Received: 7 March 1996 / Accepted: 13 March 1996

Abstract Von Willebrand factor (vWF) is the largestprotein found in plasma. It circulates in blood as a se-ries of multimers ranging in size from 500 to 20 000kDa. The variable molecular weight of vWF is due todifferences in the number of subunits comprising theprotein. vWF mediates platelet adhesion to subendo-thelium of the damaged blood vessel. Only the largestmultimers are hemostatically active. Each vWF subunitcontains binding sites for collagen and for platelet gly-coproteins GPIb and GPIIb/IIIa. Multiple interactionsof repeating binding sites in vWF multimers with adhe-sive protein(s) of the subendothelium and with recep-tors on the platelet surface lead to “irreversible” bind-ing of platelets to the exposed subendothelium. Func-tional properties of vWF are typical of multisubunitproteins encoded by autosomal loci. The phenotype ofvon Willebrand disease is determined by the propertiesof the dysfunctional subunits which become incorpo-rated into heteropolymeric forms of vWF. Absence oflarge vWF multimers, seen in type 2A von Willebranddisease and in myeloproliferative disorders, is asso-ciated with bleeding tendency. On the other hand, inpatients with vWF multimers of supranormal size, asthey occur in thrombotic thrombocytopenic purpura(TTP) and hemolytic uremic syndrome (HUS), there isan increased risk of thrombosis. Proteolytic enzyme(s)are involved in physiologic regulation of the polymericsize of vWF. We have purified from human plasma aprotease cleaving vWF at the same peptide bond that isalso cleaved in vivo. vWF was quite resistant againstthe protease in a physiologic buffer but was degradedat low salt concentration or in the presence of 1 M urea.It appears that a conformational change in the vWF

molecule exposes the specific protease-sensitive pep-tide bond and thus enhances degradation of vWF mul-timers. In some variants of type 2A vWF, the cleavagesite in the vWF subunit is more susceptible to proteo-lytic degradation than in normal vWF, whereas in pa-tients with TTP or HUS the protease activity may besuppressed. vWF-degrading protease plays an impor-tant role in pathogenesis of congenital or acquired dis-orders of hemostasis and thrombosis.

Key words von Willebrand factor 7 von Willebranddisease 7 Binding affinity 7 Multimers 7 Phenotype

Introduction

Von Willebrand factor (vWF) is a multimeric plasmaglycoprotein with two distinct biological functions: itserves as the carrier for procoagulant factor VIII andprotects it from inactivation by activated protein C andfactor Xa in the circulating blood, and it mediatesplatelet adhesion to subendothelium of the damagedblood vessel. vWF and factor VIII circulate in bloodplasma as a noncovalently associated complex consist-ing of about 99% vWF and 1% factor VIII. A de-creased concentration in the level of vWF or an abnor-mality in the interaction between vWF and factor VIIIcause a shortened half-life of factor VIII in the circulat-ing blood and thus a decrease in the level of factor VIIIactivity. Binding of platelets to the exposed subendo-thelium is the initial step in the formation of a hemo-static plug. vWF deficiency or abnormality lead to vonWillebrand disease (vWD), which is now known to bethe most common inherited human bleeding disorder.Bleeding symptoms in patients with vWF are nose andgingival bleeding, bleeding from minor skin woundsand after tooth extraction, postoperative bleeding, andmenorrhagia. vWF is synthesized exclusively by vascu-lar endothelial cells and megakaryocytes. The vWFgene, consisting of about 180 kilobases and containing52 exons, is located at the tip of the short arm of chro-

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mosome 12. Since the amino acid sequence of vWF wasdetermined, one decade ago, a large number of differ-ent mutations have been discovered that cause vWDand provide important information about the relation-ship between structure and biological functions of vWF.General reviews on vWF structure and function [1, 2]and on the pathogenesis of vWD [3, 4] have been pub-lished elsewhere.

Structure and biological functions

vWF multimers are composed of a 270-kD polypeptidesubunit comprising 2050 amino acid residues. Each sub-unit contains binding sites for collagen [5–8] and forplatelet glycoproteins GPIb [9–12] and GPIIb/IIIa. Inintact blood vessels, vWF does not interact with theplatelet receptors. It is assumed that in the injuredblood vessel, the subendothelial structures become ex-posed and bind vWF; this interaction seems to induce aconformational change in vWF leading to exposure ofbinding sites for GPIb and to platelet adhesion. It hasnot yet been established with certainty which compo-nents of the subendothelial matrix interact with vWF invivo. vWF binds not only to fibrillar collagen types I,III, and VI of the vessel wall, but also to noncollage-nous components of the subendothelium [13]. Further-more, binding of vWF to hydrophobic plastic [14] orglass [15] surfaces and high shear stress [16] wereshown to “activate” the GPIb binding site in vWF. Theinteraction of vWF with the positively charged antibiot-ic ristocetin [17] or reduction of the negative charge byremoval of sialic acid residues from the carbohydrateside-chains of vWF also result in exposure of the GPIb-binding site [18]. Botrocetin, a protein isolated from thevenom of certain pit vipers, particularly Bothrops jara-raca, was shown to bind to vWF in proximity to theGPIb binding site and to promote platelet agglutinationin the presence of vWF [19, 20]. The direct binding ofasialo-vWF to unstimulated platelets provides evidencethat GPIb receptor is normally exposed on the mem-brane of the resting platelet. Binding of vWF to GPIbinduces platelet activation, resulting in expression ofthe complex GPIIb/IIIa on the platelet membrane [21–23]. “Activated” GPIIb/IIIa is capable of cross-linkingadjoining platelets via bridges made by vWF, fibrin-(ogen), fibronectin, vitronectin, and other proteinscontaining the Arg-Gly-Asp sequence.

Size and activity of vWF

vWF circulates in plasma as a series of multimers con-taining a variable number of subunits. vWF is the lar-gest known protein present in human plasma. Flexiblestrands of up to 2 mm [24] are comparable in length tothe diameter of a medium platelet. The largest multi-mers may be as large as 20000 kD. Other large plasmaproteins with Mr`500 kD also consist of multiple poly-

Fig. 1 Subunit composition of von Willebrand factor. Electro-phoresis of normal nonreduced plasma vWF in SDS-1% agarosegel (left panel) separates vWF multimers according to their size.The sample was applied on top of the gel. After electrophoresis,the proteins were electrotransferred to nitrocellulose and thebands of vWF were detected using peroxidase-labeled rabbit IgGagainst human vWF. The bottom band, having the most rapidanodic mobility, corresponds in size to the dimer (MWF500 kD)of the vWF polypeptide subunit (middle panel). Each slower mi-grating band is one dimer larger than the faster one. The arrow(right panel) schematically illustrates the decrease of the function-al activity (binding affinity for collagen or platelet receptor GPIb)in small molecular forms of vWF

peptide subunits or of repeating domains, e.g., hapto-globin 2-2 (Mr up to 1000 kD), IgM (Mrp971 kD), a2-macroglobulin (Mrp725 kD), C4-binding protein(Mrp540 kD), and apolipoprotein B-100 (Mrp514 kD), but their molecular size is more than one or-der of magnitude smaller than that of the largest vWFmultimers. The multimeric pattern of vWF seen on so-dium dodecyl sulphate (SDS)/agarose electrophoresisshows regularly spaced bands, suggesting that eachmultimer differs from the adjacent ones by a constantmolecular mass of about 500 kD [25, 26]. Multimer for-mation takes place in the endoplasmic reticulum: ini-tially, dimers are assembled from pairs of polypeptidesubunits via disulfide bridges between cysteine residueslocated in the carboxy terminal regions; subsequently,multimers are formed by interdimeric disulfide linkingof amino terminal domains [27].

Only the large multimeric forms of vWF are hemo-statically active. In patients with bleeding disorders,both the amount and the quality of vWF have to beexamined. The functional activity of vWF is measuredby the conventional ristocetin assay, in which agglutina-tion of formalin-fixed normal human platelets by vWFpresent in patient plasma is determined turbidimetrical-ly. This platelet-agglutinating property of vWF is de-noted as ristocetin cofactor activity. The size distribu-tion of vWF is analyzed by agarose gel electrophoresisin the presence of SDS. Multimers can be visualizedwithin the gel by radiolabeled antibodies, or they canbe detected by immunoblotting techniques using en-zyme-labeled anti-vWF antibodies. Single bands appearin 1% agarose gels (Fig. 1); each band can be furtherresolved into a number of satellite bands (triplets, quin-

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tuplets) by SDS-electrophoresis in gels of higher aga-rose concentration (2–3% agarose gels). It has beenshown by gel filtration of cryoprecipitate on large-poreagarose that the large multimers eluting in the void vol-ume have a considerably higher ristocetin cofactor ac-tivity per unit antigen than the later-eluting vWF spe-cies of smaller size [28]. Gentle disulfide reduction oflarge vWF multimers resulted in decreasing molecularsize and loss of ristocetin cofactor activity; small molec-ular forms of vWF were adsorbed onto colloidal goldgranules and thereby increased their platelet-aggluti-nating activity [29]. vWF multimers of different sizeswere fractionated by gel filtration and the resultingfractions subjected to binding studies in the presence ofristocetin (vWF binding to GPIb) or after addition ofthrombin (vWF binding to the complex GPIIb/IIIa).These studies showed up to ten times higher Kd valuesfor smaller molecular forms of vWF than for largermultimers [30]. Ristocetin-induced agglutination ofwashed platelets as well as thrombin-induced plateletaggregation in platelet-rich plasma were significantlyfaster with large multimers than with small molecularforms of vWF. These investigations further support theview that the affinities for platelet receptors (GPIb andGPIIb/IIIa) of vWF are related to its multimeric size.

Multivalent binding of polymeric vWF to platelets and

collagen

Adhesion of platelets from flowing blood to collagenmay occur in the absence of vWF. Three candidate re-ceptors for collagen have been postulated: GPIa-IIa(also called VLA-2), GPIV (denoted also as CD36),and GPVI. However, a decreased level or an abnormal-ity of vWF results in reduced platelet adhesion to sub-endothelium under conditions of high shear stress andin prolonged bleeding time, indicating that direct inter-actions of exposed subendothelium with platelets viaspecific collagen receptors are insufficiently strong toresist remarkable shear forces encountered in the circu-lating blood, particularly in small vessels and stenosedarteries. The importance of the multimeric structure forfunction of vWF was confirmed by binding studies us-ing vWF of different molecular sizes. A straight linewas obtained in the Scatchard plot for interaction ofhuman fibrillar collagen type III with the dimeric pro-teolytic vWF fragment SpIII consisting of two N-termi-nal polypetide remnants (residues 1–1365), and the cal-culated association constant Ka was found [6] to be inthe range between 1.5 and 3.0!10P6 MP1. Similarbinding affinity (Kap0.5–1.0!10P6 MP1) was reportedfor binding of the monomeric proteolytic fragment SpI(residues 911–1365) [7] or recombinant vWF-A3-do-main (residues 908–1111) [8] to calf skin type I collag-en. On the other hand, Scatchard plot for binding ofpolymeric vWF to collagen showed binding behaviortypical of multiple classes of binding sites [6] with anapparent high affinity Ka of 1–3!10P8 MP1. Also the

Fig. 2 Binding of multivalent vWF to GPIb receptors on plateletsurface. A highly simplified sequence of binding steps is shown,assuming appropriate spacing of receptors with regard to the sep-aration distance between proximal functional groups. Singlybound ligand can either dissociate with rate-constant kP1 or bindto a second receptor with rate-constant k2. Doubly bound ligandcan return to its singly bound state or become triply bound.Equilibrium constants for these interactions K1`K2FK3 are pos-tulated

monomeric glycosylated recombinant vWF-A1-domain(residues 475–709) had about 100 times lower affinityfor GPIb (Kap0.22!10P6 MP1) [11] than the poly-meric human vWF [30, 31]. These data show that themultimeric structure is not absolutely essential for in-teraction of vWF with collagen or with platelet recep-tors, but the presence of repeating subunits in eachmultimer may contribute to considerably stronger in-teractions required for stable adhesion of platelets tosubendothelium in the flowing blood. A model of vWFbinding to GPIb receptors situated on the platelet sur-face is depicted in Fig. 2. The starting state is shown onthe left: multivalent ligand (vWF) in solution binds toreceptors with forward and reverse rate constants k1

and kP1, respectively. The association constant isK1pk1/kP1 and the resulting ratio of the bound to free

ligand concentration can be written as 1 rF21

p n1 K1

1cK1 F.

Singly bound vWF multimer can bind to a second re-ceptor with rate constant k2. The latter constant de-pends upon concentration of receptors at the properdistance, capable of establishing a bond with the adja-cent vWF subunit. About 20000–25000 copies of GPIbare present on the surface of a human platelet [32].Thus, the platelet surface is only sparsely covered withGPIb receptors that appear to be randomly dispersedin the platelet membrane and do not move duringplatelet activation [33]. Since the second equilibriumconstant K2 depends on appropriate spacing of plateletsurface receptors, it will presumably be significantlysmaller than K1. Theoretically, the bound/free fraction

of the doubly bound ligand is 1 rF22

pn2 K1 K2

1cK1 K2 F.

Binding of the third functional site to the third receptorwill proceed with a rate constant similar to that forbinding of the singly bound vWF to the second recep-

tor, resulting in K3;K2 and 1 rF23

;n3 K1 K2

2

1cK1 K22 F

. Final-

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ly, in a mixture of vWF multimers of variable size (com-posed of up to i dimers), the fraction of bound vWF

will be approximately rF

;i

Aip1

ni K1 KiP12

1cK1 KiP12 F

. Even if

the contribution of each subsequent equilibrium con-stant K2 is small in comparison to the initial associationconstant K1, additional bonds established by multipleinteractions of repeating binding sites with surface re-ceptors may dramatically increase the stability of thecomplex. Sufficient number of bonds represents the “ir-reversible” binding between vWF multimers and plate-lets.

Proteolytic degradation of vWF

The largest multimers are present in storage granulesboth in platelets (a-granules) and in endothelial cells(Weibel-Palade bodies), from which they are releasedinto circulation [34]. Lower molecular weight forms ofvWF in circulating blood were found to contain in-creased amounts of proteolytically cleaved fragments[35]. It has been shown that the satellite bands repre-sent proteolytic degradation products of vWF in whichterminal dimeric subunits have been cleaved by a pro-teolytic enzyme [36, 37]. It has been suggested, on thebasis of the N-terminal amino acid sequence of the cir-culating degradation fragments of vWF, that the pep-tide bond Tyr842–Met843 is cleaved by a calpain-likeprotease [38]. It appears that the size distribution ofvWF multimers may be regulated by proteolytic de-gradation of circulating vWF. If the proteolytic degrad-ation of vWF is too fast, as in some congenital variantsof vWF [39, 40] or in some myeloproliferative disorders[41], the consequence is defective hemostasis. On theother hand, in patients with extremely large vWF mul-timers, as they occur in thrombotic thrombocytopenicpurpura (TTP) [42], there is an increased thrombotictendency. It remains to be investigated whether the pa-thogenesis of the latter disorder is associated with a de-ficiency or abnormality of a protease capable of de-grading very large vWF multimers to smaller molecularforms. Our preliminary experiments have shown an im-paired activity of the vWF-degrading protease in somepatients suffering from TTP or HUS (unpublished ob-servation).

We have isolated from human plasma a vWF-cleav-ing protease using affinity chromatography and gel fil-tration [43]. An estimated purification factor of about10000 has been achieved. The proteolytic activity wasfound to be associated with a protein of MrF300 kD.High-molecular-weight vWF was apparently resistantagainst degradation when incubated with the purifiedenzyme preparation in a physiologic buffer, but it be-came degraded at low salt concentration or in the pres-ence of 1 M urea (Fig. 3). It appears that low ionicstrength or urea affect the conformation of the vWFmolecule and thus expose the cleavage site. No degrad-ation of three other proteins (human fibrinogen, bovine

Fig. 3 Influence of low ionic strength and urea on proteolytic de-gradation of von Willebrand factor. A purified preparation ofvWF was incubated with purified protease in a physiologic buffer(0.15 M NaCl), in 5 mM Tris buffer (in the absence of NaCl), atphysiologic ionic strength in the presence of urea (0.15 MNaClP1 M urea) and at low ionic strength in the presence of urea(5 mM TrisP1 M urea). Digested material was submitted to SDS-electrophoresis in agarose gel and vWF was detected by immuno-blotting using peroxidase-labeled IgG against human vWF

serum albumin, and calf skin collagen) by the purifiedprotease was observed under the same experimentalconditions. This suggests that the protease possesseshigh specificity for vWF. Proteolytic activity had a pHoptimum at 8–9 and was not affected by serine enzymeinhibitors or sulfyhdryl reagents. Inhibition by chelat-ing agents was best reversed by barium ions. The ob-served properties of the vWF-degrading enzyme differfrom those of all hitherto described proteases. Analysisof degraded vWF showed that the peptide bond Tyr842–Met843 had been cleaved – the same bond that had beenproposed to be cleaved in vivo [38].

Pathogenesis and classification of von Willebrand

disease

vWD, the most common congenital bleeding disorderin humans, with a prevalence of about 1%, is the conse-quence of quantitative and/or qualitative defects ofvWF. All vWD is caused by mutations at the vWF lo-cus. Type 1 vWD refers to partial quantitative deficien-cy of vWF and is the most frequent form of the disease,accounting for about 70% of all cases. Type 2 vWD ref-ers to qualitative deficiency of vWF. Type 3 vWD ischaracterized by the virtual absence of detectable vWFin plasma and platelets and has a prevalence of approx-imately 1 in 1000000 subjects.

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Type 2 vWD comprises four different subtypes andis phenotypically very heterogeneous. Common to allsubtypes of type 2 vWD is the occurrence of qualitativeabnormalities of vWF resulting, in most cases, in abnor-mal multimeric structure of the molecule. vWD var-iants with decreased platelet-dependent function, whichis attributed to the lack of hemostatically effective largevWF multimers, are classified under type 2A. In the la-boratory, these variants are recognized by a dispropor-tionately low value of functional vWF level comparedwith the values of the vWF antigen. Most type 2A vWDis due either to impaired secretion of large multimericforms of vWF or to enhanced proteolytic degradationof the large vWF multimers. To investigate the molecu-lar mechanism responsible for the absence of large mul-timers in type 2A vWD, processing of recombinantvWF with different known type 2A mutations has beeninvestigated. Impaired secretion of high-molecular-weight multimers was observed in variants Ser850 ]Pro [44], Gly742 ] Arg, Ser743 ] Leu, Val844 ] Asp[45], and Leu777 ] Pro [46], whereas secretion of largemultimers similar to wild-type vWF was noted withGly742 ] Glu, Arg834 ] Trp [45], and Ile865 ] Thr [46].The above mutations are located within the stretch ofamino acid residues 742–865. Apparently, all of thesemutations perturb a sensitive structure in the vWF mol-ecule, and the resulting structural alterations interferewith the intracellular polymerization and/or transport,or they result in increased susceptibility of vWF to pro-teolytic degradation at the peptide bond Tyr842–Met843.Recombinant type 2A mutants Arg834 ] Trp andArg834 ] Gln were rapidly degraded by the purifiedvWF-degrading protease in a buffer of physiologic ionicstrength in the absence of urea, while the wild-typevWF was hardly affected under the same experimentalconditions (data not shown). These results confirm thatmutations in the A2 domain may lead to an enhancedproteolytic sensitivity of vWF.

The absence of high-molecular-weight multimers isusually observed also in type 2B vWD, but this defi-ciency is caused by increased binding affinity of variantvWF for platelet receptors GPIb, resulting in an accel-erated clearance of the most adhesive, largest vWFmultimers from plasma. At least 13 missense mutationshave been identified that are linked to type 2B vWD[47]. Most of them are clustered within a short se-quence 540–578, a segment of the vWF A1 domain con-taining the putative inhibitor of vWF binding to GPIb[12]. Enhanced platelet agglutination at low ristocetinconcentration provides a fairly specific screening assayto identify vWD type 2B [48].

Type 2M vWD refers to qualitative variants with de-creased binding to platelets that is not caused by theabsence of high-molecular-weight multimers. Type 2NvWD is due to failure of vWF to bind and protect pro-coagulant factor VIII; it is characterized by normal lev-el and normal multimeric pattern of vWF but decreasedlevel of factor VIII in the circulating blood. A revisedclassification of vWD that is based on differences in pa-

thophysiology has recently been recommended [49,50].

Genotype and phenotype of vWF

Type 1 vWD is often dominant, type 2 vWD may bedominant or recessive, and type 3 vWD is recessive.Quantitative deficiencies are caused by deletions, pro-moter mutations, nonsense mutations, and frame-shiftmutations, rarely by missense mutations, whereas quali-tative defects are usually associated with missense mu-tations and small in-frame deletions and insertions.

Diagnosis of mild vWD frequently poses a problem,especially in type 1 vWD, a dominantly inherited bleed-ing disorder with apparently normal structure and func-tion of vWF coded for by the normal allele. On the oth-er hand, inheritance of a single null vWF allele is notassociated with bleeding symptoms in most heterozy-gous persons. It is possible that the numerous polymor-phisms established in the vWF gene influence the syn-thesis and function of vWF, thus affecting the pheno-type of vWD. Furthermore, the genotype at other loci,e.g., AB0 blood type and Lewis locus, has been foundto be associated with the level of plasma vWF [51, 52].vWF is increased in various stressful situations such asphysical exercise, inflammation, and pregnancy. There-fore, repeated testing of functional and antigenic levelsof vWF in plasma may be required to establish the cor-rect diagnosis of vWD.

If one allele fails to produce a functional protein, itmay be expected that vWF levels would be about 50%of normal. However, most families with type 2A vWDshow a clear dominant pattern, frequently with func-tional vWF levels of less than 10% of normal. SincevWF is multimeric, mutant subunits that are highly sus-ceptible to proteolytic degradation become incorpo-rated into vWF multimers (Fig. 4). The heterozygouscarriers of the protease-sensitive vWF are usually com-pletely devoid of large multimers, and not only of theabnormal fraction, since the functionally relevant high-molecular-weight vWF is a protease-sensitive hetero-multimer. Thus, mutation in one allele responsible forincreased protease sensitivity of vWF dominantly af-fects the phenotype of vWD.

It is conceivable that dysfunctional subunits codedby the abnormal allele produce heterodimers with nor-mal subunits via disulfide linking of the carboxy termi-nal domains (Fig. 5). The resulting dimers are dysfunc-tional, in the sense that they cannot be assembled intomultimers and secreted. This tentative model may ex-plain the dominant inheritance of the type 2A vWDthat is characterized by secretion from endothelial cellsof abnormally small molecular forms of vWF. The pres-ence of a defective “polymerization site” in only onehalf of vWF subunits is sufficient for complete deficien-cy of the very large, highly adhesive vWF multimers.Abnormal dimer polymerization may also be responsi-ble for failure of 1-deamino-8-D-arginine vasopressin

346

Fig. 4 Increased proteolytic degradation of vWF multimers intype 2A vWD. The vWF subunit consists of several structural do-mains (panel A). The platelet-binding site and one collagen-bind-ing site are located in the A1 domain. Another collagen-bindingsite is situated in the A3 domain. Extremely slowly, the proteasecleaves the peptide bond Tyr842–Met843 (white arrow) in the nor-mal vWF subunit. In an abnormal subunit (panel B), the sensitiv-ity to proteolytic cleavage is increased due to enhanced exposureof the cleavage site (black arrow). The proteolytic degradation ofvWF multimers is accelerated (panel C), since they are composedof both resistant and sensitive subunits

Fig. 5 Abnormal polymerization of dimers in type 2A vWD.Dimers are formed from normal subunits (white symbols) and ab-normal subunits (black symbols), with a mutation preventing in-terdimeric linking of the amino terminal domains. In a heterozy-gous carrier of the defect, 25% each of normal and abnormal ho-modimer will be produced, together with 50% of mixed hetero-dimer. Abnormal homodimer cannot polymerize further. The lin-ear growth of vWF multimers will be blocked following incorpo-ration of a heterodimer on each end of the polymeric chain

(DDAVP) to correct the bleeding symptoms in type 2AvWD, since predominantly small molecular forms ofvWF are released in response to DDAVP treatment.

Most amino acid substitutions established in the pro-tease-sensitive vWF variants and in abnormally poly-merizing vWF variants are situated within the samestretch of the amino acid sequence denoted as the A2

domain. Mutations within the same codon resulting inGly742 ] Arg and Gly742 ] Glu have even been shownto result in either decreased processing or increasedproteolytic breakdown of highly polymeric vWF, re-spectively [45]. It is conceivable that various mutationsin the A2 domain may affect the multimeric distribu-tion by both impaired secretion of vWF multimers andenhanced sensitivity to the plasma protease.

Similar considerations might be applied to the prop-erties of the type 2B vWF variants with increased affin-ity for GPIb. In heterozygotes, large polymers contain-ing both normal and abnormal subunits will exhibit anincreased binding affinity for platelet receptors. Themode of inheritance of type 2B vWD will be dominant,due to spontaneous adsorption of heteropolymericvWF to platelets. In contrast to types 2A and 2B vWD,the functional defect in type 2N vWD is not dependenton multimeric size: procoagulant factor VIII binds as amonovalent ligand to vWF. The abnormal binding offactor VIII is not affected by formation of heteropo-lymeric forms, and the trait is inherited in a recessivemanner. DDAVP is relatively ineffective in patientswith type 2N; its therapeutic effect may be of only ashort duration.

In spite of significant progress in our understandingof the synthesis, structure, and functions of vWF, thevariable presentation of bleeding symptoms in patientswith vWD remains poorly understood. Due to the highprevalence of vWD, there is a high probability of com-pound heterozygosity. Although the new classificationhelps in predicting the response to DDAVP, the thera-peutic efficacy of DDAVP will often require appro-priate testing of individual patients.

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